Glycoprotein hormone long-acting superagonists

ABSTRACT

This invention provides long-acting, superactive analogs of glycoprotein hormones demonstrating enhanced bioactivity both in vitro and in vivo as compared to wild type counterparts. The analogs are particularly useful for treating subjects showing low receptor expression or poor receptor responsiveness, and for the treatment of any condition associated with glycoprotein hormone activity.

SEQUENCE LISTING INFORMATION

A computer readable text file, entitled“056815-5010-SequenceListing.txt,” created on or about Nov. 20, 2013with a file size of about 30 kb contains the sequence listing for thisapplication and is hereby incorporated by reference in its entirety.

FIELD OF INVENTION

This invention relates generally to modified glycoprotein hormoneshaving superagonist activity, and the use thereof in the treatment ofconditions associated with glycoprotein hormone activity. Morespecifically, this invention relates to modified glycoprotein moleculescontaining amino acid substitutions and one or more inserted peptides inthe alpha subunit as compared to wild type alpha subunit, wherein suchmodified molecules exhibit enhanced pharmacological properties ascompared to wild type glycoproteins.

BACKGROUND OF INVENTION

The gonadotropins follitropin (follicle-stimulating hormone, FSH) andchorionic gonadotropin, (CG), lutropin (luteinizing hormone, LH), andthyrotropin (thyroid-stimulating hormone, TSH) comprise the family ofglycoprotein hormones. Each hormone is a heterodimer of twonon-covalently linked subunits: alpha and beta. Within the same species,the amino acid sequence of the alpha-subunit is identical in all thehormones, whereas the sequence of the beta-subunit is hormone specific(Pierce, Ann. Rev. Biochem. 50:465-495 (1981)). The fact that thesequences of the subunits are highly conserved from fish to mammalsimplies that these hormones have evolved from a common ancestral protein(Fontaine, Gen. Comp. Endocrinol. 32:341-347 (1977)).

Previous studies with modified glycoprotein hormones have revealedencouraging data. For example, in addition to providing modifiedglycoprotein hormones with increased activity, further mutations havedemonstrated an increase in receptor affinity binding (see e.g. WO2005/089445 and WO 2005/101000). However, while affinity was increased,studies demonstrated that modified glycoprotein hormones were cleared asquickly if not faster than their wild type counterparts. In order togenerate a clinically useful superagonist with enhanced activity,modified glycoprotein superagonist should have an improved biologicalhalf-life in addition to improved receptor binding affinity. However,previous attempts to further modify glycoprotein hormones to increasehalf-life and improve bioavailability have been less than satisfactoryand instead the modified glycoprotein hormones demonstrated only anattenuated response.

SUMMARY OF INVENTION

The invention encompasses a modified glycoprotein hormone comprising anamino acid sequence with at least one conservative basic amino acidsubstitution at Q13, E14, P16 or Q20 and an insert of VNVTINVT (SEQ IDNO: 20) between D3 and Q5 of the alpha subunit of a glycoproteinhormone.

In some embodiments, the modified glycoprotein hormone comprises atleast two or at least three basic amino acid substitutions at Q13, P16and Q20. In some embodiments, the modified glycoprotein hormone furthercomprises a basic amino acid substitution at E14. In some embodiments,the basic amino acid is arginine.

In some embodiments, the alpha subunit comprises an amino acid sequencewith at least 85% identity to SEQ ID NO: 11 and further comprises thebeta subunit of leutenizing hormone (LH), chorionic gonadotropin (CG),follicle-stimulating hormone (FSH) or thyroid-stimulating hormone (TSH).In some embodiments, the alpha subunit is derived from a human alphasubunit (SEQ ID NO: 6).

The invention encompasses a modified glycoprotein hormone comprising anamino acid sequence with at least one conservative basic amino acidsubstitution at K15, K17, K20 or K24 and an insert of NVTINV (SEQ IDNO: 1) between F6 and T7 of the alpha subunit of a glycoprotein hormone.

In some embodiments, modified glycoprotein hormone comprises at leasttwo, or at least three, or at least four basic amino acid substitutionsat K15, K17, K20 and K24. In some embodiments, the modified glycoproteinhormone further comprises a basic amino acid substitution at E18. Insome embodiments, the basic amino acid is arginine.

In some embodiments, the alpha subunit comprises an amino acid sequencewith at least 85% identity to SEQ ID NO: 7 and further comprises thebeta subunit of leutenizing hormone (LH), chorionic gonadotropin (CG),follicle-stimulating hormone (FSH) or thyroid-stimulating hormone (TSH).In some embodiments, the alpha subunit is derived from a bovine,porcine, or ovine alpha subunit (SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO:3, respectively).

The invention encompasses a modified glycoprotein hormone comprising anamino acid sequence with at least one conservative basic amino acidsubstitution at K15, E18, K20 or K24 and an insert of NVTINV (SEQ IDNO: 1) between F6 and T7 or alternatively an insert of NV between F6 andT7 plus an insert of INV between T7 and T8 of the alpha subunit of aglycoprotein hormone.

In some embodiments, modified glycoprotein hormone comprises at leasttwo, or at least three, or at least four basic amino acid substitutionsat K15, E18, K20 and K24. In some embodiments, the modified glycoproteinhormone includes an insert of NVTINV (SEQ ID NO: 1) between F6 and T7 ofthe alpha subunit. In some embodiments, the modified glycoproteinhormone includes an insert of NV between F6 and T7 plus an insert of INVbetween T7 and T8 of the alpha subunit. In some embodiments, the basicamino acid is arginine of histidine. In some embodiments, the basicamino acid is arginine.

In some embodiments, the alpha subunit comprises an amino acid sequencewith at least 85% identity to SEQ ID NO: 4 and further comprises thebeta subunit of leutenizing hormone (LH), chorionic gonadotropin (CG),follicle-stimulating hormone (FSH) or thyroid-stimulating hormone (TSH).In some embodiments, the alpha subunit is derived from an equine alphasubunit (SEQ ID NO: 4).

The invention also encompasses a method for stimulating a glycoproteinreceptor in an animal, comprising administering of the above modifiedglycoprotein hormones to the animal. The invention also encompasses amethod for stimulating ovulation in an animal comprising administeringany of the above the modified glycoprotein hormones to the animal. Insome embodiments the animal is a human, cow, sheep, pig or horse.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows cAMP stimulation in CHO-FSHR cells with selected bFSHanalogs produced by transient transfection. FIG. 1A shows a comparisonof Folltropin®-V (pFSH), bFSH-WT (wild-type) with bFSH-5R analog. FIG.1B shows attenuation of the in vitro bioactivity of hFSH-TR4402 analog(Transient 4402) by two N-terminal extensions (ANITV, NITV) and oneinternal neoglycosylation (V78N) (SPA cAMP assay). FIG. 1C shows acomparison of bFSH-5R analog with the Insert 1 (5R+Insert 1) and Insert2 (5R+Insert2).

FIGS. 2A and 2B show PK screening assay of various bFSH analogs aftersingle subcutaneous injection in mice. In each experiment, 5 mice wereused for each preparation. Blood samples were taken at 24, 32 and 48 hafter injection, plasma levels were deducted and the data were expressedas % of injected dose (% ID). FSH levels in plasma samples were assayedusing FSH ELISA (Endodrine Technologies).

FIGS. 3A-D show the analysis of different lots of TR55601 production.FIG. 3A illustrates charge heterogeneity analysis using IEF followed byWestern blot. Suboptimal sialylation of Lot 3 (Lanes 2 and 3) is insharp contrast with optimal highly acidic isoforms detected in Lot 4(Lanes 5 and 6). Lane 1, IEF 3-10 marker; Lane 2, TR55601/Lot 3 (8 μg);Lane 3, TR55601/Lot 3 (4 μg); Lane 4 and 8, TR4401 (1 μg); Lane 5,TR55601/Lot 4 (8 μg); Lane 6, TR55601/Lot 4 (4 μg); Lane 8, IEF 3-10marker. FIG. 3B illustrates Analysis of charged isoforms usingneuraminidase (Vibrio cholerae), IEF and Western blot. UntreatedTR55601/Lot 4 sample (Lanes 2 and 3) and TR55601/Lot 4 sample treatedwith neuraminidase before applying to 3-10 IEF gel (Lanes 4-6). Lane 1,IEF 3-10 marker; Lane 2, untreated TR55601/Lot 4 (8 μg); Lane 3,untreated TR55601/Lot 4 (4 μg); Lane 4, treated TR55601/Lot 4 (4 μg);Lane 5, treated TR55601/Lot 4 (2 μg); Lane 6, treated TR55601/Lot 4 (1μg). The IEF profile for the neuraminidase digested isoforms has shiftedto the pI range from 7.8 to 10.0. The average shift of pI is about 5 pHunits and multiple bands (close to 10 bands) transformed into one majorband (pI˜9.5) and three minor bands (pI 7.8-10.0), indicating that themajority of the observed charge heterogeneity (FIG. 3A—Lot 4) isdependent on terminal sialic acid residues with a minor component ofother modifications such as deamidation and/or proteolytic degradation.The residual basic bands (pI 4.8-5.5) shown in 3B are nonspecific,derived from neuraminidase preparation. FIG. 3C shows the analysis ofcharged isoforms of TR55601-Lot 5 by IEF 3-10 in pI gradient gel (IEF3-10) followed by Western blotting. Lane 1, IEF 3-10 marker; Lane 2,TR55601-Lot 5 (4 μg); Lane 3, TR55601-Lot 5 (4 μg); Lane 4, TR55601-Lot4 (4 μg); Lane 5, TR55601-Lot 4 (8 μg); Lane 6, TR55601-Lot 3 (4 μg);lane 7, TR55601-Lot 3 (8 μg). FIG. 3D illustrates SDS-Western Blotanalysis of TR55601Lot 5 compared to TR55601Lot 4 and Fol-V. Lane 1,protein marker; Lane 2: Lot 4, 500 ng; Lane 3: Lot 5, 1 ul; Lane 4:empty lane; Lane 5: Lot 4, 4 ug; Lane 6: Lot 5, 15 ul; Lane 7: Fol-V,673 ng; Lane 8: protein marker.

FIG. 4 shows the results from Classic Steelman-Pohley bioassay with hCGaugmentation of ovarian weight in immature (22 days old) Sprague-Dawleyfemale rats. Ovarian weights were measured 72 hours after dosing. Dataare presented as average total ovarian weight of two ovaries+SEM (n=5per dose, per group). Rats were stimulated with one single injection oftest article or vehicle, supplemented with 40 IU of hCG. The followingdosage groups were used: Group 1 was receiving hCG only (no FSH), Groups2-5 were receiving TR55601Lot 4 (0.33 μg, 1.0 μg, 3.33 μg, and 10 μg,respectively from left to right), Groups 6-8 were receivingFolltropin-V® (3,333 μg, 10,000 mg, and 30,000 μg respectively from leftto right), and Group 9-10 was receiving TR4401 (1.0 mg and 3.33 μg).

FIG. 5 demonstrates a follicular wave synchronizing protocol forsuperovulation, induction of ovulation and fixed-time artificialinsemination. The 8 injections of Folltropin-VR (Bioniche) are replacedwith a single or double injection of TR55601.

FIG. 6 shows the mean number of follicles (3 to 5 mm in diameter) duringsuperstimulation treatment in beef cows treated with 60 μg rFSH given bya single I.M. injection or 300 mg Folltropin-V (Control) given in twicedaily I.M. injections over 4 days (3 experiments combined).

FIG. 7 shows the mean number of follicles 6 to 8 mm in diameter duringsuperstimulation treatment in beef cows treated with 60 μg rFSH given bya single I.M. injection or 300 mg Folltropin-V (Control) given in twicedaily I.M. injections over 4 days (3 experiments combined).

FIG. 8 shows the mean number of follicles >9 mm in diameter duringsuperstimulation treatment in beef cows treated with 60 μg rFSH given bya single I.M. injection or 300 mg Folltropin-V (Control) given in twicedaily I.M. injections over 4 days (3 experiments combined).

FIG. 9 shows the mean diameter profiles of all follicles ≧3 mm indiameter during superstimulation treatment in beef cows treated with 60μg rFSH given by a single I.M. injection or 300 mg Folltropin-V(Control) given in twice daily I.M. injections over 4 days (3experiments combined).

FIG. 10A shows a comparison in cAMP production for the insert in thehuman alpha subunit (A2), the insert without the amino-terminal valine(Insert 2), the 5 arginine substitutions only without the insert (5R)and control of medium only. FIG. 10B shows the EC50 for the threeconstructs tested.

FIG. 11A shows a comparison of cAMP production in response to the humanmodified alpha subunit with the insert of SEQ ID NO: 1 and the bovinemodified alpha subunits that lack the insert. FIG. 11B shows acomparison of cAMP production in response to the human modified alphasubunit and the bovine subunits with and without various inserts.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides modified superactive glycoprotein hormonemolecules showing surprisingly enhanced potency and increased biologicalhalf-life as compared to their wild type counterparts. Being modifiedmeans that, while the protein contains an amino acid sequence whichdiffers from the wild-type glycoprotein hormones, the sequence has beenchanged such that it is not identical to the known glycoprotein hormonessequence of another species. Superactivity may be assessed according toa variety of parameters, including potency and efficacy. Potency is aparameter of bioactivity that is determined by measuring the halfmaximal response. Differences in potency are determined by comparing thevalue of the glycoprotein hormones response of the analog halfwaybetween baseline and maximum (EC₅₀) versus that of wild typeglycoprotein hormones. Glycoprotein hormone responses may be measured invitro using purified proteins, or may be estimated following transienttransfection of a nucleic acid encoding the modified protein.Glycoprotein hormone responses may also be measured in vivo, i.e. in ananimal responsive to said glycoprotein hormone analog. Such responsesencompass any known cellular or biological and quantitative orqualitative response of glycoprotein hormone binding to its receptor,e.g. cAMP production, synthesis of proteins such as progesterone,fertilization rate, blastocyst formation rate, embryo development perfertilized oocyte, etc. Efficacy (Vmax) or maximum response is anotherparameter of bioactivity. As discussed herein, parameters of bioactivitymay vary depending on receptor number and receptor coupling in the assaycell line. In systems with lower receptor numbers or impaired coupling,differences are more discernable in terms of Vmax (efficacy). In systemswhere receptors are overexpressed, differences in potency are morevisible.

For example, in instances where the modified glycoprotein hormone is amodified FSH or CG molecule, in vivo quantitative and qualitativeparameters such as quantity of oocytes, fertilization rate andblastocyst and embryo formation rates may be measured at the maximallyeffective dose for oocyte number. The maximally effective dose foroocyte number is the optimal amount of superactive FSH for both oocytequality and quantity. The maximally effective dose for oocyte number isdependent on an animal's weight and rate of metabolism. For example, themaximally effective dose for a larger animal with a slower rate ofmetabolism is greater than the maximally effective dose for a smalleranimal with a higher rate of metabolism. The maximally effective dose isdetermined empirically for each animal.

However, regardless of the system used, the modified superactiveglycoprotein hormone proteins of the invention may demonstrate at leastabout a 2 to 10 fold increase in potency or at least about a 20-fold,30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold or even100-fold increase in potency compared to a wild type counterpart, orabout a 2 to 10% increase in maximal efficacy, or at least a 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, or even 100% increase in maximal efficacycompared to a wild type counterpart. The superactive analogs of theinvention may also provide about a five to ten fold increase in potencyor 5% to 10% increase in maximal efficacy as compared to wild type FSH.Some of the modified proteins of the invention may demonstrate at leastabout a thirty to fifty fold increase in potency or 30% to 50% increasein maximal efficacy as compared to wild type. Thus, the modifiedglycoprotein hormone proteins of the present invention may be useful fortreating subjects with low receptor number or deficiencies in receptorresponse, since the modified proteins of the invention may maintain atleast a 10 fold increase in potency or 10% increase in maximal efficacyeven in systems with low receptor number or response.

The rate of absorption of a modified superactive glycoprotein hormonemay result in increased duration of action. A modified glycoproteinhormone analog with a decreased rate of absorption and increasedduration of action may be beneficial for hyposensitive subjects, such asthose suffering from fertility disorders. The rate of absorption ismeasured by K_(a). The rate of elimination is measured by K_(e).

The modified glycoprotein hormone molecules of the invention includemodified proteins of species selected from the group consisting ofhuman, bovine, equine, porcine, ovine, murine, rat, rabbit, primate,etc. Fish glycoprotein hormone (also known as GTH-1) may be used inaquaculture, i.e., in order to assist growth of endangered or other fishspecies in captivity. Other species of modified glycoprotein hormonesmay be used in agricultural breeding, and further in a laboratorysetting for testing the effects of different combined mutations onvarious male and female glycoprotein hormone-related conditions.

The modified glycoprotein hormone molecules of other species havesubstitutions at positions corresponding to those in the modified human(e.g. FIG. 12), bovine (see FIG. 4), ovine, equine and porcineglycoprotein hormone molecules disclosed herein, which may be identifiedusing any alignment program, including but not limited to DNASIS,ALIONment, SIM and GCG programs such as Gap, BestFit, FrameAlign andCompare.

Modified glycoprotein hormone molecules of the present inventioncomprise at least a modified alpha-subunit, wherein the alpha subunitcomprises at least two basic amino acids such at lysine residues. Inhuman alpha subunits, the basic amino acids may be introduced atpositions 13, 14, 16 and 20 of wild type human alpha subunit (SEQ ID NO:6). In other species, the basic amino acids may be introduced atpositions corresponding to positions 15, 17, 20 and 24 of wild typebovine alpha (SEQ ID NO: 2), wild type porcine alpha (SEQ ID NO: 5) andwild type ovine alpha (SEQ ID NO: 3), and positions 15, 20 and 24 ofwild type equine alpha (SEQ ID NO: 4). The glutamate residue at position18 (bovine, porcine, ovine and equine) may also be substituted with abasic amino acid. In some embodiments, the basic amino acid may bearginine or histidine. In some embodiments, the basic amino acid may bearginine.

A peptide with the sequence NVTINV (SEQ ID NO: 1) or TNVTINV (SEQ ID NO:12) or VNVTINVT (SEQ ID NO: 20) may be inserted between amino acids D3and Q5 of the human alpha subunit (SEQ ID NO: 6) and between F6 and T7of the bovine, porcine, ovine and equine alpha subunits. Alternatively,modified glycoprotein hormone alpha subunit of bovine, porcine, ovine oreuqine may include an insert of NV between F6 and T7 plus an insert ofINV between T7 and T8. The modified proteins of the invention may alsocontain further substitutions, particularly conservative substitutionsthat do not alter the enhanced properties of the protein. Typically,however, such modified proteins will contain less than fivesubstitutions at positions other than those listed above, and mayexhibit complete amino acid sequence identity with the correspondingwild-type glycoprotein hormone alpha in positions other than thepositions listed above.

Basic amino acids comprise the amino acids lysine, arginine, andhistidine, and any other basic amino acid which may be a modification toany of these three amino acids, synthetic basic amino acids not normallyfound in nature, or any other amino acid which is positively charged ata neutral pH. The basic amino acids, among others, are selected from thegroup consisting of lysine and arginine.

Exemplary modified alpha molecules having the basic amino acidsubstitutions and the peptide insert are set forth in SEQ ID NO: 11(human), SEQ ID NO: 7 (bovine), SEQ ID NO: 8 (ovine), SEQ ID NO: 10(porcine), and SEQ ID NO: 9 (equine). The present invention provides formodified glycoproteins with amino acid sequences with at least 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% orhigher identity with any of SEQ ID NO: 7 to 11.

The modified alpha subunits of the modified glycoprotein hormoneproteins of the invention may also have an alpha subunit comprising two,three, four or five basic amino acid substitutions. The substitutedamino acids may be lysine residues, glutamate residues, proline residuesor glutamine residues. For example, in wild type bovine alpha subunits,one or more of the lysines at positions 15, 17, 20 and 24 may besubstituted, as well as the glutamate at position 18 with a basic aminoacid, such as an arginine and a histidine. In wild type human alphasubunits, one or more of the glutamines at positions 13 and 20 may besubstituted, as well as the glutamate at position 14 and the proline atposition 16 with a basic amino acid, such as an arginine and ahistidine. In wild type porcine alpha subunits, one or more of thelysines at positions 15, 17, 20 and 24 may be substituted, as well asthe glutamate at position 18 with a basic amino acid, such as anarginine and a histidine. In wild type ovine alpha subunits, one or moreof the lysines at positions 15, 17, 20 and 24 may be substituted, aswell as the glutamate at position 18 with a basic amino acid, such as anarginine and a histidine. In wild type equine alpha subunits, one ormore of the lysines at positions 15, 20 and 24 may be substituted, aswell as the glutamate at position 18 with a basic amino acid, such as anarginine and a histidine.

By way of example, further modified bovine alpha subunits are presentedin the sequences as set forth in SEQ ID NO: 14 to 19 and 22. The presentinvention provides for modified glycoproteins with amino acid sequenceswith at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or higher identity with any of SEQ ID NO: 14 to 19and 22.

By way of example, further modified equine alpha subunits are presentedin the sequences as set forth in SEQ ID NO: 38 to 42. The presentinvention provides for modified glycoproteins with amino acid sequenceswith at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or higher identity with any of SEQ ID NO: 43 to 45.

Further modified alpha subunits may be designed by comparing the aminoacid sequences of the alpha subunit of interest to that of other speciesto identify the corresponding basic residues in the proteins of otherspecies. Such methods are disclosed in U.S. Pat. No. 6,361,992 which isherein incorporated by reference in its entirety. Consideration may alsobe given to the relative biological activity of the glycoprotein hormonefrom various species as to which species to choose for comparison andsubstitution. Further, homology modeling based on the structure ofrelated glycoprotein hormones is useful to identify surface-exposedamino acid residues. To modify additional amino acid positions,glycoprotein hormone sequences from human and non-humans can be alignedusing standard computer software programs such as DNASIS (HitachiSoftware Engineering) or any of the other alignment programs listedabove, including but not limited to ALIONment, SIM and GCG programs suchas Gap, BestFit, FrameAlign and Compare. The amino acid residues thatdiffer between the human and the non-human glycoprotein hormone can thenbe substituted using one of the above-mentioned techniques, and theresultant glycoprotein hormone assayed for its potency using one of theherein-mentioned assays.

Accordingly, the present invention also provides a modified FSH proteinhaving increased potency over a wild-type FSH from the same speciescomprising the modified alpha subunits described herein.

The present invention also provides a modified LH protein havingincreased potency over a wild-type LH from the same species comprisingthe modified alpha subunits described herein.

The present invention also provides a modified TSH protein havingincreased potency over a wild-type TSH from the same species comprisingthe modified alpha subunits described herein.

The present invention also provides a modified CG protein havingincreased potency over a wild-type CG from the same species comprisingthe modified alpha subunits described herein.

The present invention also encompasses fragments of the analogsdescribed herein that have either superagonist or antagonist activity.For example, fragments of the modified alpha chains of the invention maybe used either alone or in combination with either a fragment or fulllength beta chain to create superagonist compounds. In some cases,fragments of the modified alpha subunit molecules of the invention mayalso be used as antagonists, for instance, to limit the duration ofactivity of an glycoprotein hormone therapeutic after it has beenadministered.

The present invention also provides for nucleic acid sequences encodingthe modified glycoprotein hormones described herein. The presentinvention also provides nucleic acids that encode polypeptides withconservative amino acid substitutions. The nucleic acids of the presentinvention may encode polypeptides that transport sugar. The isolatednucleic acids may have at least about 30%, 40%, 50%, 60%, 70%, 80% 85%,90%, 95%, or 99% sequence identity with the above identified sequences.The isolated nucleic acids may encode a polypeptide having an amino acidsequence having at least about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%,95%, or 99% sequence identity to amino acid sequences encoded by theabove identified accession numbers. The isolated nucleic acid encoding atransporter may hybridize to the above identified nucleic acidsequences.

The nucleic acid encoding the modified glycoprotein hormone proteins maybe genetically fused to expression control sequences for expression.Suitable expression control sequences include promoters that areapplicable in the target host organism. Such promoters are well known tothe person skilled in the art for diverse hosts from prokaryotic andeukaryotic organisms and are described in the literature. For example,such promoters may be isolated from naturally occurring genes or may besynthetic or chimeric promoters.

The present invention also provides expression cassettes for insertingthe nucleic acid encoding a modified glycoprotein hormone protein intotarget nucleic acid molecules such as vectors. For this purpose, theexpression cassette is provided with nucleotide sequences at the 5′- and3′-flanks to facilitate removal from and insertion into specificsequence positions like, for instance, restriction enzyme recognitionsites or target sequences for homologous recombination as, e.g.catalyzed by recombinases. In addition to the nucleic acid molecule orexpression cassette of the invention, the vector may contain furthergenes such as marker genes which allow for the selection of said vectorin a suitable host cell and under suitable conditions. Generally, thevector also contains one or more origins of replication. The vectors mayalso comprise terminator sequences to limit the length of transcriptionbeyond the nucleic acid encoding the transporters of the presentinvention.

Advantageously, the nucleic acid molecules contained in the vectors areoperably linked to expression control sequences allowing expression,i.e. ensuring transcription and synthesis of a translatable RNA, inprokaryotic or eukaryotic cells.

The term isolated refers to molecules separated from other cell/tissueconstituents (e.g. DNA or RNA), that are present in the natural sourceof the macromolecule. The term isolated as used herein also refers to anucleic acid or peptide that is substantially free of cellular material,viral material, and culture medium when produced by recombinant DNAtechniques, or that is substantially free of chemical precursors orother chemicals when chemically synthesized. Moreover, an isolatednucleic acid or peptide may include nucleic acid or peptide fragmentswhich are not naturally occurring as fragments and would not be found inthe natural state.

The terms plasmid and vector are used interchangeably as the plasmid isthe most commonly used form of vector. However, the invention isintended to include such other forms of expression vectors which serveequivalent functions and which become known in the art subsequentlyhereto. A vector may be any of a number of nucleic acids into which adesired sequence may be inserted by restriction and ligation fortransport between different genetic environments or for expression in ahost cell. Vectors are typically composed of DNA, although RNA vectorsare also available. Vectors include, but are not limited to, plasmidsand phagemids. A cloning vector is one which is able to replicate in ahost cell, and which is further characterized by one or moreendonuclease restriction sites at which the vector may be cut in adeterminable fashion and into which a desired DNA sequence may beligated such that the new recombinant vector retains its ability toreplicate in the host cell. In the case of plasmids, replication of thedesired sequence may occur many times as the plasmid increases in copynumber within the host bacterium or just a single time per host beforethe host reproduces by mitosis. In the case of phage, replication mayoccur actively during a lytic phase or passively during a lysogenicphase.

Vectors may further contain a promoter sequence. A promoter may includean untranslated nucleic acid sequence usually located upstream of thecoding region that contains the site for initiating transcription of thenucleic acid. The promoter region may also include other elements thatact as regulators of gene expression. In further embodiments of theinvention, the expression vector contains an additional region to aid inselection of cells that have the expression vector incorporated. Thepromoter sequence is often bounded (inclusively) at its 3′ terminus bythe transcription initiation site and extends upstream (5′ direction) toinclude the minimum number of bases or elements necessary to initiatetranscription at levels detectable above background. Within the promotersequence will be found a transcription initiation site, as well asprotein binding domains responsible for the binding of RNA polymerase.Eukaryotic promoters will often, but not always, contain TATA boxes andCAT boxes. Activation of promoters may be specific to certain cells ortissues, for example by transcription factors only expressed in certaintissues, or the promoter may be ubiquitous and capable of expression inmost cells or tissues.

Vectors may further contain one or more marker sequences suitable foruse in the identification and selection of cells which have beentransformed or transfected with the vector. Markers include, forexample, genes encoding proteins which increase or decrease eitherresistance or sensitivity to antibiotics or other compounds, genes whichencode enzymes whose activities are detectable by standard assays knownin the art (e.g., β-galactosidase or alkaline phosphatase), and geneswhich visibly affect the phenotype of transformed or transfected cells,hosts, colonies or plaques. Vectors may be those capable of autonomousreplication and expression of the structural gene products present inthe DNA segments to which they are operably joined. An expression vectoris one into which a desired nucleic acid sequence may be inserted byrestriction and ligation such that it is operably joined or operablylinked to regulatory sequences and may be expressed as an RNAtranscript. Expression refers to the transcription and/or translation ofan endogenous gene, transgene or coding region in a cell.

A coding sequence and a regulatory sequence are operably joined whenthey are covalently linked in such a way as to place the expression ortranscription of the coding sequence under the influence or control ofthe regulatory sequences. If it is desired that the coding sequences betranslated into a functional protein, two DNA sequences are said to beoperably joined if induction of a promoter in the 5′ regulatorysequences results in the transcription of the coding sequence and if thenature of the linkage between the two DNA sequences does not (1) resultin the introduction of a frame-shift mutation, (2) interfere with theability of the promoter region to direct the transcription of the codingsequences, or (3) interfere with the ability of the corresponding RNAtranscript to be translated into a protein. Thus, a promoter regionwould be operably joined to a coding sequence if the promoter regionwere capable of effecting transcription of that DNA sequence such thatthe resulting transcript might be translated into the desired protein orpolypeptide.

Some aspects of the present invention include the transformation and/ortransfection of nucleic acids. Transformation is the introduction ofexogenous or heterologous nucleic acid to the interior of a prokaryoticcell. Transfection is the introduction of exogenous or heterologousnucleic acid to the interior of a eukaryotic cell. The transforming ortransfecting nucleic acid may or may not be integrated (covalentlylinked) into chromosomal DNA making up the genome of the cell. Inprokaryotes, for example, the transforming nucleic acid may bemaintained on an episomal element such as a plasmid or viral vector.With respect to eukaryotic cells, a stably transfected cell is one inwhich the transfecting nucleic acid has become integrated into achromosome so that it is inherited by daughter cells through chromosomereplication. This stability is demonstrated by the ability of theeukaryotic cell to establish cell lines or clones comprised of apopulation of daughter cells containing the transfected nucleic acid.

There are numerous E. coli (Escherichia coli) expression vectors knownto one of ordinary skill in the art which are useful for the expressionof the nucleic acid insert. Other microbial hosts suitable for useinclude bacilli, such as Bacillus subtilis, and otherenterobacteriaceae, such as Salmonella, Serratia, and variousPseudomonas species. In these prokaryotic hosts one can also makeexpression vectors, which will typically contain expression controlsequences compatible with the host cell (e.g., an origin ofreplication). In addition, any number of a variety of well-knownpromoters will be present, such as the lactose promoter system, atryptophan (Trp) promoter system, a beta-lactamase promoter system, or apromoter system from phage lambda. The promoters will typically controlexpression, optionally with an operator sequence, and have ribosomebinding site sequences for example, for initiating and completingtranscription and translation. If necessary, an amino terminalmethionine can be provided by insertion of a Met codon 5′ and in-framewith the downstream nucleic acid insert. Also, the carboxyl-terminalextension of the nucleic acid insert can be removed using standardoligonucleotide mutagenesis procedures.

Additionally, yeast expression can be used. There are several advantagesto yeast expression systems. First, evidence exists that proteinsproduced in a yeast secretion systems exhibit correct disulfide pairing.Second, post-translational glycosylation is efficiently carried out byyeast secretory systems. The Saccharomyces cerevisiaepre-pro-alpha-factor leader region (encoded by the MF″-1 gene) isroutinely used to direct protein secretion from yeast. (Brake, Proc.Nat. Acad. Sci., 81:4642-4646 (1984)). The leader region ofpre-pro-alpha-factor contains a signal peptide and a pro-segment whichincludes a recognition sequence for a yeast protease encoded by the KEX2gene: this enzyme cleaves the precursor protein on the carboxyl side ofa Lys-Arg dipeptide cleavage signal sequence. The FSH coding sequencecan be fused in-frame to the pre-pro-alpha-factor leader region. Thisconstruct is then put under the control of a strong transcriptionpromoter, such as the alcohol dehydrogenase I promoter or a glycolyticpromoter. The nucleic acid coding sequence is followed by a translationtermination codon which is followed by transcription terminationsignals. Alternatively, the nucleic acid coding sequences can be fusedto a second protein coding sequence, such as Sj26 orbeta.-galactosidase, which may be used to facilitate purification of thefusion protein by affinity chromatography. The insertion of proteasecleavage sites to separate the components of the fusion protein isapplicable to constructs used for expression in yeast. Efficientpost-translational glycosylation and expression of recombinant proteinscan also be achieved in Baculovirus systems.

Mammalian cells permit the expression of proteins in an environment thatfavors important post-translational modifications such as folding andcysteine pairing, addition of complex carbohydrate structures, andsecretion of active protein. Vectors useful for the expression of activeproteins in mammalian cells are characterized by insertion of theprotein coding sequence between a strong viral promoter and apolyadenylation signal. The vectors can contain genes conferringhygromycin resistance, gentamicin resistance, or other genes orphenotypes suitable for use as selectable markers, or methotrexateresistance for gene amplification. The chimeric protein coding sequencecan be introduced into a Chinese hamster ovary (CHO) cell line using amethotrexate resistance-encoding vector, or other cell lines usingsuitable selection markers. Presence of the vector DNA in transformedcells can be confirmed by Southern blot analysis. Production of RNAcorresponding to the insert coding sequence can be confirmed by Northernblot analysis. A number of other suitable host cell lines capable ofsecreting intact human proteins have been developed in the art, andinclude the CHO cell lines, HeLa cells, myeloma cell lines, Jurkatcells, etc. Expression vectors for these cells can include expressioncontrol sequences, such as an origin of replication, a promoter, anenhancer, and necessary information processing sites, such as ribosomebinding sites, RNA splice sites, polyadenylation sites, andtranscriptional terminator sequences. Exemplary expression controlsequences are promoters derived from immunoglobulin genes, SV40,Adenovirus, Bovine Papilloma Virus, etc. The vectors containing thenucleic acid segments of interest can be transferred into the host cellby well-known methods, which vary depending on the type of cellularhost. For example, calcium chloride transformation is commonly utilizedfor prokaryotic cells, whereas calcium phosphate, DEAE dextran, orlipofectin mediated transfection or electroporation may be used forother cellular hosts.

Alternative vectors for the expression of genes in mammalian cells,those similar to those developed for the expression of humangamma-interferon, tissue plasminogen activator, clotting Factor VIII,hepatitis B virus surface antigen, protease Nexin1, and eosinophil majorbasic protein, can be employed. Further, the vector can include CMVpromoter sequences and a polyadenylation signal available for expressionof inserted nucleic acids in mammalian cells (such as COS-7).

Expression of the gene or hybrid gene can be by either in vivo or invitro. In vivo synthesis comprises transforming prokaryotic oreukaryotic cells that can serve as host cells for the vector.Alternatively, expression of the gene can occur in an in vitroexpression system. For example, in vitro transcription systems arecommercially available which are routinely used to synthesize relativelylarge amounts of mRNA. In such in vitro transcription systems, thenucleic acid encoding the glycoprotein hormone would be cloned into anexpression vector adjacent to a transcription promoter. For example, theBluescript II cloning and expression vectors contain multiple cloningsites which are flanked by strong prokaryotic transcription promoters(Stratagene). Kits are available which contain all the necessaryreagents for in vitro synthesis of an RNA from a DNA template such asthe Bluescript vectors (Stratagene). RNA produced in vitro by a systemsuch as this can then be translated in vitro to produce the desiredglycoprotein hormone (Stratagene).

Another method of producing a glycoprotein hormone is to link twopeptides or polypeptides together by protein chemistry techniques. Forexample, peptides or polypeptides can be chemically synthesized usingcurrently available laboratory equipment using either Fmoc(9-fluorenylmethyloxycarbonyl) or Boc (tert-butyloxycarbonoyl) chemistry(Applied Biosystems). One skilled in the art can readily appreciate thata peptide or polypeptide corresponding to a hybrid glycoprotein hormonecan be synthesized by standard chemical reactions. For example, apeptide or polypeptide can be synthesized and not cleaved from itssynthesis resin whereas the other fragment of a hybrid peptide can besynthesized and subsequently cleaved from the resin, thereby exposing aterminal group which is functionally blocked on the other fragment. Bypeptide condensation reactions, these two fragments can be covalentlyjoined via a peptide bond at their carboxyl and amino termini,respectively, to form a hybrid peptide. (Grant, Synthetic Peptides: AUser Guide, W.H. Freeman (1992) and Bodansky, Principles of PeptideSynthesis, Springer-Verlag (1993)). Alternatively, the peptide orpolypeptide can by independently synthesized in vivo as described above.Once isolated, these independent peptides or polypeptides may be linkedto form a glycoprotein hormone via similar peptide condensationreactions. For example, enzymatic or chemical ligation of cloned orsynthetic peptide segments can allow relatively short peptide fragmentsto be joined to produce larger peptide fragments, polypeptides or wholeprotein domains (Abrahmsen, Biochemistry, 30:4151 (1991); Dawson,Science, 266:776-779 (1994)).

The modified glycoprotein hormones of the present invention can berecombinant proteins obtained by cloning nucleic acids encoding thepolypeptide in an expression system capable of producing the polypeptidefragments thereof. For example, one can determine the active domain of amodified alpha subunit which, together with the beta subunit, caninteract with a glycoprotein hormone receptor and cause a biologicaleffect associated with the glycoprotein hormone. In one example, aminoacids found to not contribute to either the activity or the bindingspecificity or affinity of the glycoprotein hormone can be deletedwithout a loss in the respective activity.

For example, amino or carboxyl-terminal amino acids can be sequentiallyremoved from either the native or the modified glycoprotein hormone andthe respective activity tested in one of many available assays describedabove. In another example, the modified proteins of the invention mayhave a portion of either amino terminal or carboxyl terminal aminoacids, or even an internal region of the hormone, replaced with apolypeptide fragment or other moiety, such as biotin, which canfacilitate in the purification of the modified glycoprotein hormone. Forexample, a modified glycoprotein can be fused to a maltose bindingprotein, through either peptide chemistry of cloning the respectivenucleic acids encoding the two polypeptide fragments into an expressionvector such that the expression of the coding region results in a hybridpolypeptide. The hybrid polypeptide can be affinity purified by passingit over an amylose affinity column, and the modified glycoprotein canthen be separated from the maltose binding region by cleaving the hybridpolypeptide with the specific protease factor Xa.

Active fragments of the modified glycoprotein hormone molecules of theinvention can also be synthesized directly or obtained by chemical ormechanical disruption of larger glycoprotein hormone. An active fragmentis defined as an amino acid sequence of at least about 5 consecutiveamino acids derived from the naturally occurring amino acid sequence,which has the relevant activity, e.g., binding or regulatory activity.The fragments, whether attached to other sequences or not, can alsoinclude insertions, deletions, substitutions, or other selectedmodifications of particular regions or specific amino acids residues,provided the activity of the peptide is not significantly altered orimpaired compared to the modified glycoprotein hormone. Thesemodifications can provide for some additional property, such as toremove/add amino acids capable of disulfide bonding, to increase itsbio-longevity, etc. In any case, the peptide must possess a bioactiveproperty, such as binding activity, regulation of binding at the bindingdomain, etc. Functional or active regions of the glycoprotein hormonemay be identified by mutagenesis of a specific region of the hormone,followed by expression and testing of the expressed polypeptide. Suchmethods are readily apparent to a skilled practitioner in the art andcan include site-specific mutagenesis of the nucleic acid encoding thereceptor.

The present invention also encompasses fusion proteins and chimericproteins comprising the mutations described herein, including forinstance, fusions to the FSH glycoprotein. Such a fusion protein may bemade by ligating the appropriate nucleic acid sequences encoding thedesired amino acid sequences to each other by methods known in the art,in the proper coding frame, and expressing the fusion protein by any ofthe means described above. Alternatively, such a fusion protein may bemade by protein synthesis techniques, for example, using a peptidesynthesizer. The single chain analogs and chimeric proteins of theinvention may incorporate a peptide linker between the alpha and betasubunits, or between different portions of the chimeric protein.

Characterization of Glycoprotein Hormone Superagonists

The effect of the modification or modifications to the wild-typeglycoprotein hormones described herein can be ascertained in any numberof ways. For example, changes to second messenger systems within cellstransfected with a nucleic acid encoding the modified glycoproteinhormones can be measured and compared to similar cells transfected witha nucleic acid encoding the wild-type glycoprotein hormone.Alternatively, the activity of a modified glycoprotein hormone can bedetermined from receptor binding assays, from thymidine uptake assays,from progesterone production assays, or from T4 secretion assays. Oneskilled in the art can readily determine any appropriate assay to employto determine the activity of either a wild-type or a modifiedglycoprotein hormone.

In one embodiment of the present invention, the modified glycoproteinhormone has a potency which is increased over the potency of the wildtype glycoprotein hormone. This increased potency can be assessed by anyof the techniques mentioned above or in any other appropriate assay asreadily determined by one skilled in the art. The increased potency doesnot have to be consistent from assay to assay, or from cell line to cellline, as these of course, will vary.

In another embodiment of the present invention, the modifiedglycoprotein hormone has a maximal efficacy which is increased over themaximal efficacy of the wild type glycoprotein hormone. This increasedmaximal efficacy can be assessed by any of the techniques mentionedabove or in any other appropriate assay as readily determined by oneskilled in the art. The increased maximal efficacy does not have to beconsistent from assay to assay, or from cell line to cell line, as theseof course, will vary.

Other assays suitable for characterizing the analogs described hereinare described in PCT/US99/05908, which is herein incorporated byreference in its entirety. For instance, various immunoassays may beused including but not limited to competitive and non-competitive assaysystems using techniques such as radioimmunoassays, ELISA, Isoelectricfocusing (IEF) assays, sandwich immunoassays, immunoradiometric assays,gel diffusion precipitin reactions, immunodiffusion assays, in situimmunoassays, western blots, precipitation reactions, agglutinationassays, complement fixation assays, immunofluorescence assays, protein Aassays, and immunoelectrophoresis assays, etc.

For example, when the beta subunit is that of FSH, improvements in thequality and quantity of oocytes can be assessed by in vitro and in vivoassays. Superactive FSH can be used to improve the quality and quantityof oocytes from animals, including but not limited to, human, mouse,rat, primate, rabbit, pig, horse, sheep, and dog. Preferably, asuperactive FSH is administered to a human or any animal. It is commonfor improvements in oocyte quantity and quality to be determined usingdifferent end points of the in vitro fertilization process such asoocyte formation, oocyte fertilization, and blastocyst formation. Invitro fertilization experiments may follow a “superovulation protocol”in which subjects are treated with a superactive FSH analog according tothe present invention, which leads to the release and maturation ofmultiple oocytes. In in vitro fertilization experiments, FSH(superactive FSH and recombinant wild type FSH) may be administered withhCG to trigger ovulation. A control animal may be used which receivesonly hCG or pregnant mare serum gonadotropin (PMSG). The quality ofoocytes can be improved by increasing the fertilization rate of oocytesin an animal. The fertilization rate of a superactive folliclestimulating hormone can be determined in vivo or in vitro by comparingthe fertilization rate achieved with a superactive FSH to thefertilization rate achieved with the same amount of recombinant wildtype FSH. A control animal may also be used that receives hCG. The rateof fertilization can be measured by the percent of two-cell embryoswhich develop per total number of oocytes. If fertilization takes placein vitro, two cell embryos can be counted in fertilization dishes. Inmice, two cell embryos develop approximately twenty-four hours afterfertilization. The fertilization rate varies based on the amount ofsuperactive FSH administered. An animal may receive multiple does ofsuperactive FSH. The rate of fertilization increases by at least about10 percent as a result of administration of superactive FSH at themaximally effective dose for oocyte number. The rate of fertilizationmay increase by at least about 20 percent, preferably at least 30%, 40%,50%, 60%, 70%, 80%, 90%, or 100% as a result of administration ofsuperactive FSH at the maximally effective dose for oocyte number.Superactive follicle stimulating hormone can improve the quality ofoocytes by improving the blastocyst formation rate per fertilizedoocyte. The rate of blastocyst formation can be measured by determiningthe percentage of two-cell embryos which form blastocysts. The rate ofblastocyst formation increases whether the blastocyst forms in vivo orin vitro. The blastocyst formation rate is dependent on the amount ofsuperactive follicle stimulating hormone administered. The rate ofblastocyst formation increases at least about 10 percent as a result ofadministration of a superactive follicle stimulating hormone at themaximally effective dose for oocyte number. The rate of blastocystformation may increase at least about 20 percent, preferably at least30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% as a result of administrationof superactive FSH at the maximally effective dose for oocyte number.

Superactive follicle stimulating hormone can improve the quality ofoocytes by increasing the total number of embryos per fertilized oocyte.The increase in total number of embryos per fertilized oocyte increaseswhether fertilization occurs in vivo or in vitro. The increase in totalnumber of embryos per fertilized oocyte is dependent on the amount ofsuperactive follicle stimulating hormone administered. The total numberof embryos per fertilized oocyte increases at least about 10 percent asa result of administration of a superactive follicle stimulating hormoneat the maximally effective dose for oocyte number. The total number ofembryos per fertilized oocyte may increase by at least about 20 percent,preferably at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% as aresult of administration of superactive FSH at the maximally effectivedose for oocyte number.

For example, when the beta subunit is that of CG, potent luteinizinghormone (LH)-like activities can be assessed by in vitro and in vivobioassays. Superactive CG induces ovulation, extends the life span ofcorpus luteum, increases progesterone synthesis and promotes theformation of accessory corpora lutea in certain species. Such actionsresult in more effective oocyte collection, increase in oocyte qualityin certain species, increase of pregnancy and pregnancy maintenancerates.

Glycoprotein Hormone Analogs with Increased Serum Half-Life

The modified glycoprotein hormone proteins of the invention may also befurther modified such that the plasma half-life is increased as comparedto wild type counterparts. The modified glycoprotein hormone proteins ofthe invention may further comprise a potential glycosylation siteincluding sequences comprising N-glycosylation and/or O-glycosylationsites. For example, placement of the peptide NVTINV (SEQ ID NO: 1) orVNVTINVT (SEQ ID NO: 20) in the alpha subunit provides for potentialglycosylation site of the alpha subunit. The peptides of SEQ ID NO: 20or SEQ ID NO: 1 may be placed in the human wild type sequences betweenD3 and Q5. The peptides of SEQ ID NO: 20 or SEQ ID NO: 1 may be placedin the bovine, equine, porcine and ovine wild type sequences between F6and T7. The inserted peptide may further comprise an additionalthreonine residue at the amino terminus. Further peptides to insert inorder to alter glycosylation include NV, INV, and TNV peptides, as wellas TNVTINV (SEQ ID NO: 12). For example, a modified alpha subunit ofglycohormone may include an insert of NV between F6 and T7 plus aninsert of INV between T7 and T8. Increased half-life may also beprovided by pegylation or conjugation of other appropriate chemicalgroups or by constructing fusion proteins having increased half life orany other method. Such methods are known in the art, for instance asdescribed in U.S. Pat. No. 5,612,034, U.S. Pat. No. 6,225,449, and U.S.Pat. No. 6,555,660, each of which is incorporated by reference in itsentirety.

Half-life may also be increased by increasing the number of negativelycharged residues within the molecule, for instance, the number ofglutamate and/or aspartate residues. Such alteration may be accomplishedby site directed mutagenesis. Such alteration may also be achieved viaan insertion of an amino acid sequence containing one or more negativelycharged residues into the modified glycoprotein hormone proteins.

The half-life of a protein is a measurement of protein stability andindicates the time necessary for a one-half reduction in theconcentration of the protein. The serum half-life of the modifiedglycoprotein hormone proteins described herein may be determined by anymethod suitable for measuring hormone levels in samples from a subjectover time, for example but not limited to, immunoassays using antibodiesto measure levels in serum samples taken over a period of time afteradministration of the modified glycoprotein hormone proteins, or bydetection of labeled hormone molecules, i.e., radiolabeled molecules, insamples taken from a subject after administration of the labeledglycoprotein hormones.

Methods of Treatment

The modified glycoprotein hormone proteins of the present invention maybe used to treat any condition associated with glycoprotein hormoneactivity. The modified glycoprotein hormone proteins of the presentinvention may be used to treat a subject in need thereof. A subject maybe an animal, such as a mammal, a reptile, a fish, a bird and anamphibiab. The subject may be a mammal, such as a human, cow, sheep, pigor horse. An animal includes livestock and domesticated pets, such ascanines and felines. The subject may be a human patient or an animal inneed of improved glycoprotein hormone activity. Conditions associatedwith glycoprotein hormone activity are ones that are either completelyor partially caused by altered glycoprotein hormone responsiveness, orones that benefit from the administration of glycoprotein hormone. Forinstance, such conditions include, but are not limited to ovulatorydysfunction, luteal phase defects, unexplained infertility, male factorinfertility, time-limited conception, low FSH receptor expression, lowFSH receptor sensitivity, FSH receptor binding deficiencies, FSHreceptor coupling deficiencies, low testosterone production, malepattern baldness, and pituitary failure or injury.

For example, the quantity and quality of oocytes can be improved byadministering a superactive FSH analog as described herein to an animal.For example, Applicants have surprisingly found that by administering asuperactive FSH containing a modified alpha-subunit, a dramatic increasein the quantity and quality of oocytes is obtained. The effects of asuperactive FSH on oocyte quantity and quality may be further enhancedby increasing the FSH serum half-life of the superactive FSH. The FSHserum half-life can be increased by further modifying the superactiveFSH. Further modifications, including but not limited to thosepreviously described, can be used to increase FSH serum half-life.

The modified FSH, CG, LH, or TSH glycoprotein hormone proteins of thepresent invention may also be used in therapeutic regimens of assistedreproduction in either a male or female subject comprising administeringan assisting amount of the modified glycoprotein hormone proteins to thesubject. In such methods, the analogs may be administered alone or incombination with other therapeutics, for instance, including but notlimited to Clomiphene citrate and GnRH (gonotropin releasing hormone).The modified glycoprotein hormone proteins of the present invention maybe administered as a combination of one or more glycoproteins. Forexample, a modified alpha subunit may be combined with a FSH betasubunit, a CG beta subunit, a TSH beta subunit, and/or a LH betasubunit, together or separately, and the modified glycoproteins are thenadministered to a subject. For example, in a subject with isolatedgonadotropin deficiency (IGD), modified FSH, CG, TSH, and LH may beadministered to the subject to restore normal gonadal function. It iswidely known in the art that glycoprotein hormones such as FSH, CG, TSH,LH are integral in female reproductive physiology, and theseglycoprotein hormones may be administered to a subject to overcome anumber of reproductive disorders and thereby assist reproduction.

Single and multiple injection dosing regimens are tested for themodified equine CG glycoprotein. For superstimulation using eCG analogin horses, cows, or pigs, single or 2:1 split eCG analog injection isused. Dose ranging studies for eCG analog include a single im. injectionof 30, 45, 60, 75, 90, 105 and 120 mcg. Optimized dose are tested in 2:1ratio and the optional second split dose will coincide with PGF2alphatreatment on Day 6 or another date. The treatment with the modified eCGproduces superovalation in horses, cows, and pigs.

A skilled practitioner in the art can readily determine the effectiveamount of the glycoprotein hormone to administer and will depend onfactors such as weight, size, the severity of the specific condition,and the type of subject itself. The therapeutically effective amount canreadily be determined by routine optimization procedures. The presentinvention provides glycoprotein hormones with increased potency relativeto the wild-type glycoprotein hormone. These modified glycoproteinhormones will allow a skilled practitioner to administer a lower dose ofa modified glycoprotein hormone relative to the wild-type glycoproteinhormones to achieve a similar therapeutic effect, or alternatively,administer a dose of the modified glycoprotein hormone similar to thedose of the wild-type glycoprotein hormone to achieve an increasedtherapeutic effect.

Depending on whether the glycoprotein hormone is administered orally,parenterally, or otherwise, the administration of the prostaglandin canbe in the form of solid, semi-solid, or liquid dosage forms, such as,for example, tablets, pills, capsules, powders, liquids, creams, andsuspensions, or the like, preferably in unit dosage form suitable fordelivery of a precise dosage. The glycoprotein hormone may include aneffective amount of the selected glycoprotein hormone in combinationwith a pharmaceutically acceptable carrier and, in addition, may includeother medicinal agents, pharmaceutical agents, carriers, adjuvants,diluents, etc. By “pharmaceutically acceptable” is meant a material thatis not biologically or otherwise undesirable, i.e., the material may beadministered to an individual along with the selected glycoproteinhormone without causing unacceptable biological effects or interactingin an unacceptable manner with the glycoprotein hormone. Actual methodsof preparing such dosage forms are known, or will be apparent, to thoseskilled in this art; for example, see Remington's PharmaceuticalSciences, latest edition (Mack Publishing).

The following examples are provided to describe and illustrate thepresent invention. As such, they should not be construed to limit thescope of the invention. Those in the art will well appreciate that manyother embodiments also fall within the scope of the invention, as it isdescribed hereinabove and in the claims.

EXAMPLES Design of Alpha Subunit Analogs

A human FSH superagonist glycoprotein with modifications to theα-subunit at Q13R+E14R+P16R+Q20R (human 4R) with a wild-type β-subunitdemonstrated significant binding superiority over their wild-typecounterparts.

Table 1 shows a comparison of human alpha wild-type (WT) and selectedhFSH superagonists primary amino acid structure. N-terminal portions ofhuman alpha wild-type (amino acid residues 1-28 of 92 total residues)and mutated forms are shown. Location of 4 superagonist substitutions toarginine (R) is in the shaded area. Selected 4 different insertsintroducing one or two additional N-linked carbohydrate chains aremarked between amino acid D3 and Q5 of the wild-type sequence.

TABLE 1

The segments in Table 1 are listed as the following: SEQ ID NO: 43: hFSHWT; SEQ ID NO: 33, hFSH alpha (4R); SEQ ID NO: 34, hFSH alpha (4R+Ins1);SEQ ID NO: 35, hFSH alpha (4R+Ins2); SEQ ID NO: 36, hFSH alpha(4R+Ins3); SEQ ID NO: 37, hFSH alpha (4R+Ins4).

The bovine FSH (bFSH) substitutions in some embodiments are highlyanalogous to the residues previously mutagenized in the human FSH alphasubunit and include combination of 5 mutations called “5R”(K15R+K17R+E18R+K20R+K24R). E.g. SEQ ID NO: 7. To increase theprobability that introduced glycosylation recognition sequences (NXT orNXS) leads to attachment of N-linked carbohydrate chain, 18 differentbovine alpha subunit constructs were established and cloned intopreviously developed expression vectors. 12 constructs containedN-terminal extension peptide sequences ANITV, ANTTA, ANTSA, ANITVNITV,ANTSANTTA and ANTSANTSA.

Table 2 shows a comprison of bovine alpha wild-type (WT) and selectedbFSH superagonists primary amino acid structures. N-terminal portions ofbovine alpha wild-type (amino acid residues 1-32 of 96 total residues)and mutant bovine alpha are shown. Location of 5 superagonistsubstitutions to arginine (R) or lysine (K) are marked in the shadedarea between amino acids C14 and P25, coded as 5R of 4R+1K. Selected 4different inserts introducing one or two additional N-linkedcarbohydrate chains are marked in between amino acid F6 and T8 of thewild-type sequence.

The segments in Table 2 are listed as the following: SEQ ID NO: 44, WTbFSH; SEQ ID NO: 23, bFSH alpha (5R); SEQ ID NO: 24, bFSH alpha (4R+1K);SEQ ID NO: 25, bFSH alpha (5R+Ins1); SEQ ID NO: 26, bFSH alpha(5R+Ins2); SEQ ID NO: 27, bFSH alpha (5R+Ins3); SEQ ID NO: 28, bFSHalpha (5R+Ins4); SEQ ID NO: 29, bFSH alpha (4R+1K+Ins1); SEQ ID NO: 30,bFSH alpha (4R+1K+Ins2); SEQ ID NO: 31, bFSH alpha (4R+1K+Ins3); SEQ IDNO: 32, bFSH alpha (4R+1K+Ins4).

TABLE 2

Equine glycoprotein alpha subunits including mutations were alsoproduced and some embodiments include substitution of K15R, E18R, K2OR,and K24R. E.g. SEQ ID NO: 9; SEQ ID NO: 38-40. In addition, equineglycoprotein alpha subunits having K15R, E18H, K20R, and K24R were alsogenerated (e.g. SEQ ID NO: 41; SEQ ID NO: 42). These mutated alphasubunit also contain insert of NVTINV (SEQ ID NO: 1) between F6 and T7of the subunit (e.g. SEQ ID NO: 9, 40, and 42) or NV insert between F6and T7 plus INV between T7 and T8 (e.g. SEQ ID NO: 38, 39, and 41).

Transient transfection of bFSH analogs using polyethylenimine (PEI)resulted in 3.7-4.5 fold increase in bFSH analog expression incomparison to lipofectamine-based methods (data not shown). Expressionlevels of various FSH analogs based on heterodimer-specific ELISAindicated no major loss of FSH dimer formation and do not supportprevious claims that a single chain construct was necessary to achievehigh level expression.

Selection of transiently expressed bFSH analogs included cAMP-based invitro bioassay and PK screening assays (see FIGS. 1 and 2). There was asignificant 3-4 fold increase in potency of bFSH with 5R substitutionsin comparison to porcine FSH (pFSH-Folltropin®-V) and bFSH controls(FIG. 1A). Remarkably, in contrast to many previous studies (FIG. 1B),(Heikopp, Eur. J. Biochem 261: 81-84, 1999; Trousdale, Fertil. Steril.91: 265-270, 2009) novel neoglycosylation inserts have been found not todecrease in vitro bioactivity of bovine FSH 5R analog (FIG. 1C).Identical neoglycosylation sites added at the N-terminus reduced invitro bioactivity of bFSH, similar to the attenuating effect ofneoglycosylation on the intrinsic activity of erythropoietin (Elliott,Exp. Hematol. 32: 1146-1155, 2004; Elliott, Nat. Biotechnol. 21:4144-421, 2003; Sinclair, J. Pharm. Sci. 94: 1626-1635, 2005) and theeffect of many other prolongations of half-life approaches includingsite-directed pegylation (Fishburn, J. Pharm. Sci. 97: 4167-4183, 2008;Uchiyama, Vet. J. 184: 208-211, 2010). Two days PK screening study inmice indicated that all neo-glycosylated bovine FSH “5R” analogs hadincreased terminal half life in comparison to bFSH-WT and Folltropin®-V(FIGS. 2A and 2B). The data from the PK screening assay indicatedgreatly prolonged plasma half-life due to glycosylation at one or twointroduced neoglycosylation sites (Insert 1 and 2) in comparison tobFSH-WT, Folltropin®-V and TR4401 controls. Observed levels werecomparable with bFSH-single chain molecule with 29 amino acid linker and4-5 O-linked carbohydrate chains. Two initially tested analogs made as acombined superagonist and neoglycosylation inserts, named “Insert 1 and2” had in vitro bioactivity comparable to 5R superagonist control alone(FIG. 1C) and yet still prolonged half life in mice (FIG. 2B). Such longacting analogs without reduction of superagonist activity areunprecedented and translates to expected impressive performance in vivoin cows.

Several hundreds of milligrams of various human FSH and TSH wereproduced recombinantly (rFSH or rTSH) with CHO cells using flasks,shakers, roller bottles and bioreactors. During initial work adicistronic retroviral vector system was optimized for high levelexpression of FSH and TSH analogs in CHO-DG44 cells. Human rFSHpreparation was tested. A single 60 μg dose of rFSH induced follicledevelopment resulting in high number of good quality embryos matchingpreviously optimized eight injections of Folltropin®-V (300 mg)administered twice daily over 4 days further supporting its uniqueproperties such as delayed absorption after I.M. injection, as well asenhanced FSH receptor residency time. See FIGS. 3 and 5-8. The rFSHsuperagonist at a 10 μg dose showed exceptional ability to recruit andmaintain over 12 days enhanced pool of growing follicles, in particularfollicles in 3-5 mm size range, which are also known in humans to havelow FSH receptor number (data not shown). This unexpected enhancementand support of small follicles by the rFSH, not observed with previouslyoptimized dosing of control Folltropin®-V, provides a new way to recruitFSH responsive follicles at random stages of the cycle and enhancepotential for successful IVF and superovulation in a number of poorresponders caused by decreased FSH receptor number or function (PerezMayorga, J. Clin. Endocrinology 152: 3268-3369, 2000; Levallet, Arch.Med. Red. 30: 486-494, 1999; Rannikko, Mol. Hum. Reprod. 8: 311-317,2002; Cai, Feril. Steril. 87: 1350-1356, 2007). However, because of the40 amino acid difference between bovine FSH and human FSH based-alphasubunit with 4 arginine substitutions there were observed some carryovereffects from previous treatments with the rFSH in the same cows, whichwas in agreement with previous data study showing immunogenic propertiesof human FSH in rabbits and Rhesus monkeys (Cai, Int. J. Toxicol. 30:153-161, 2011; De Castro, Theriogenology 72: 655-662, 2009).

The introduction of arginine (R) or lysine (K) residues into a selectedmodification permissive area of the common alpha-subunit has beenpreviously shown to modulate activity of glycoprotein hormones duringevolution (Szkudlinski, Nat. Biotechnol. 14: 1257-1263, 1996;Szkudlinski, Physiol. Rev. 82: 473-502, 2002) and play an important rolein the electrostatic interaction with the negatively charged clusterlocated in the hinge region of glycoprotein hormone receptors (Mueller,Trends Endocrinol. Metab. 21: 111-122, 2010; Mueller, J. Biol. Chem.284: 16317-16324, 2009; Mueller, Endocrinol. 152: 3268-3278, 2011).Specific bFSH superagonist development includes minimal substitutions to4-5 R and/or K to produce a more potent and efficacious molecule withpossible delayed absorption to increase duration of action as shown inthe data herein and other studies for hFSH and other glycoproteinhormone analogs (Szkudlinski, Physiol. Rev. 82: 473-502, 2002). Theminimal length amino acid inserts containing one or two carbohydrateneoglycosylation sites to increase halflife to produce a singleinjection analog without reducing increased superagonistpotency/efficacy has also been investigated (see FIG. 4). For furtheranalysis, 8 constructs containing peptide inserts NVTINV, NVTINVT, NVand NVT located between amino acid 6 and 8 of the wild-type sequence canbe used. As shown in the data herein and in contrast to previousneoglycosylation and pegylation studies (Trousdale, Feril. Steril. 91:265-270, 2009; Uchiyama, Vet J. 184: 208-211, 2010; Perlman, J. Clin.Endocrinol. Metab. 88: 3227-3235, 2003) it is possible to engineerminimal length amino acid insert containing complex carbohydrate toincrease half-life without reducing increased superagonistpotency/efficacy. These novel analogs can be expressed by transienttransfection in CHO-K1 cells using the optimized high expression systemmethod using PEI and roller bottles.

Purification of selected 4-6 analogs expressed by transient transfectionmay be performed using a capture step using SP Sepharose column,followed by selecting analogs with Mono Q ion exchange chromatographybefore final polishing using gel filtration. The purity of bFSH analogscan be greater than 98%. Cumulative recovery may reach 50% with a total50-fold purification. All analogs may be characterized in vitro by ELISAimmunoassay, robust in vitro cAMP bioassay using CHO-FSHR cell line,SDS-PAGE electrophoresis and isoelectric focusing (IEF) gel analysis.Selected purified analogs may also be analyzed by rigorousquantification by reverse phase HPLC, carbohydrate compositionalanalysis and assessments of stability and aggregation states.

Further experiments produced bovine FSH analog TR55601 (alpha subunit:SEQ ID NO: 7), which includes substitutions to arginine (R) in 15K, 17K,18K, 20K, and 24K of the alpha subunit as well as an NVTINV (SEQ IDNO: 1) insert between 6F and 7T of the alpha subunit. Several lots ofTR55601 were generated and tested using IEF and Western blot analysis.FIGS. 3A-D show exemplary results of the analysis. FIGS. 3A-3Ddemonstrate, in part, that TR55601/Lot 4 and Lot 5 display optimaldistribution and verify the effectiveness of the mutations in this Lot.Lots 4 and 5 and Lots having similar screening results were used inanimal treatments. For example, the IEF-Western Blot analysis illustratethe optimized acidic isoforms of TR56001 of Lot 4 and Lot 5 in FIGS.3A-C. On the other hand, as shown in FIGS. 3A and 3C, Lot 3 was notfully verified and was not used in animal treatments. The bFSH analogsand in particular the samples of TR55601 were also studied with PKscreening assays in mice. Mice were subcutaneously injected withselected bFSH samples and blood samples were taken at 24, 32, and 48hours after injections. Plasmas were isolated and analyzed with bFSHELISA (Endocrine Technologies, Inc.). The prolonged half life of Lot 4of the TR55601 samples was confirmed with the PK screening assays. Datanot shown.

Full pharmacokinetic profile of selected candidate analogs may beperformed in by sc administration of a single dose of 10 μg per rat and10 different blood collections times (1, 5, 15, 30 min and 1, 2, 6, 24and 48 h) spanning both distribution and elimination phase. Bovine FSHplasma levels may be quantified in plasma using bFSH-ELISA. Full PKanalysis may be performed.

Analogs were selected for constructing bicistronic expression vectors,and selection and amplification of analog expression in CHO-DG44 cellsin preparation for large-scale production, purification andsuperovulation studies in cattle. The CHO-DHFR(−) DG44 cells wereco-transfected with the expression vectors and submitted to geneamplification in culture medium containing stepwise increments ofmethotrexate (MTX). Cells were qualified for the next amplification stepafter regaining their polygonal morphology (2-3 weeks). Clones thatpresent a secretion level>2 pg/cell/day may be subjected to a secondtreatment, directed to amplify the GS marker gene (MSX). An additional2-5 fold increase may be obtained, reaching a secretion level up to 10pg/cell/day.

Preparation and Experimentation with Alpha Subunit Analogs

Although the rFSH induced a superovulatory response following a singleintramuscular (I.M.) injection and 60 μg would appear to be very closeto the optimal dose, there was a significant decrease in superovulatoryresponse when cows were exposed to the rFSH for three times or more.Therefore, experiments were designed to test a new rFSH formulationcalled TR 55601 rFSH, which includes an alpha subunit (SEQ ID NO: 7)having a “5R” substitution and an insert of NVTINV (SEQ ID NO: 1)between F6 and T7. The objective was to first determine the effect of asingle or split dose treatment with rFSH to induce a superovulatoryresponse in beef cows and then to further evaluate the superovulatoryresponse of the single injection of rFSH and to determine if thesuperovulatory response remained high when cows were exposed to rFSH twoor three times.

The TR55601 FSH samples were analyzed in vivo in rats with the classicSteelman-Pohley FSH bioassay (Steelman et al., Endocrinol. 53: 604-616,1953). Female Sprague-Dawley rats (200-220 g) were injected with onesingle dose of test article (e.g. bFSH) or vehicle, supplemented with 40IU of hCG. Ovarian weights were measured 72 hours after dosing. See FIG.4. Each group is treated with hCG to provide a baseline. TR55601 bFSH,at all concentrations, significantly increases ovarian weight.

FIG. 5 shows a follicular wave synchronizing protocol forsuperovulation, induction of ovulation and fixed-time artificialinsemination, wherein the 8 injections of Folltropin-VR (Bioniche) arereplaced with a single or double injection of TR55601. Treatmentincluded insertion of progesterone (P4) releasing intravaginal deviceand administration of benzoate estradiol (BE) on Day 0. Superovulatorytreatments were initiated on Day 4 with TR55601 given as a single ordouble injection. Second split-dose injection was coinciding withPGF_(2α) treatments on Day 6. Progesterone device was removed with thelast FSH injection on Day 7. On day 8 donors were receiving porcine LHand were inseminated without estrus detection 12 and 24 h later, or onceon Day 8 (16 h after pLH). Ova/embryos were collected non-surgically onDay 15 (2, 3). Folltropin-V® was used as a control; total 300 mg* wasgiven in 8 intramuscular (IM) injections twice daily over 4 days(mg*—based on highly impure NIH-FSH-P1 Reference Standard).

In particular, 30 nonlactating Red Angus cows were stratified andblocked based on their previous history of embryo production andrandomly assigned to one of three treatment groups. Cows in the Controlgroup (n=10) received 300 mg Folltropin-V, I.M. in a twice dailydecreasing dose protocol administered over a 4-day period. Specifically:Day 4, 3.0 mL (am and pm); Day 5, 2.5 mL (am and pm); Day 6, 1.5 mL (amand pm) and Day 7, 0.5 mL (am and pm). Cows in the rFSH 60 μg treatmentgroup received a single I.M. injection of 60 μg rFSH and cows in therFSH 40-20 μg treatment group received an I.M. injection of 40 μg rFSHon Day 4, followed by another I.M. injection of 20 μg rFSH on Day 6.

Then, 25 out of the 30 cows were superstimulated again with Folltropin-V(Control) or a single injection of 60 μg of rFSH Animals in the Controlgroup (n=10) remained in the Control group, and 8 out 10 cows in therFSH 60 μg in Experiment 1 were treated again by a single I.M. injectionof rFSH. Furthermore, 7 out of 10 cows previously treated with thesplit-single injection of rFSH were treated with a single I.M. injectionof rFSH. The interval between the embryo collections was 29 days.

24 of the 25 cows used in the second experiment were superstimulatedagain with Folltropin-V (Control) or a single injection of 60 μg ofrFSH. Again, Control cows remained in the Control group and rFSH cowsremained in the rFSH group. The interval between embryo collections is30 days.

On Day 0 (beginning of experiment), all animals received 5 mgestradiol-1713 plus 50 mg progesterone and an intravaginal deviceimpregnated with progesterone (Cue-Mate, Bioniche Animal Health). On Day4 (expected day of follicle wave emergence), all cows weresuperstimulated according to the groups described above. The 60 μg dosesfor the single I.M. injection rFSH constituted 7.5 mL and Folltropin-Vwas administered in 8 I.M. injections in a decreasing dose protocol. Allanimals received 500 μg of cloprostenol I.M. (Cyclase, Syntex,Argentina) on Day 6 in the morning and in the evening. Cue-mates wereremoved in the evening of Day 6. In the morning of Day 8, cows received100 μg of gonadorelin (Gonasyn, Syntex Argentina) and inseminated 12 and24 hours later. All cows were inseminated with frozen semen from thesame bull. Ova/embryos were collected non-surgically on Day 15 andevaluated following IETS recommendations.

All cows were examined ultrasonically on Days 0, 4, 6 and 8 for thepresence of a CL and follicle size and number and to determine folliclegrowth profiles. Ovulatory response was confirmed by counting the numberof CL and follicles >10 mm in diameter by ultrasonography and rectalpalpation on Day 15.

In each experiment, data points were first evaluated for normality andhomogeneity of variance. Because variances differed among groups, datawere transformed by square root and analyzed by one-way ANOVA. Analysisof the overall response after the three experiments are concluded willbe done by two-way ANOVA to detect the effect of experiment number andtreatment and their interaction. Means were compared by the protectedLSD test. Follicle data were analyzed by the MIXED procedure to detectthe effect of treatment, day and their interaction on follicle numbersand growth profiles. All analyses were done using the InfostatAnalytical Software (Universidad Nacional de Cordoba, Argentina).

Superovulatory response and ova/embryo data are summarized in Table 3.Although the mean number of CL and cows with ≦2 CL on the day ofova/embryo collection did not differ among groups, the split-injectionof rFSH resulted in a higher (P<0.05) number of unovulated follicles.The mean number of total ova/embryos, fertilized ova and transferableembryos (Grades 1, 2 & 3) also did not differ.

Table 3 shows superovulation with single or split-single doses ofTR55601 (rFSH).

TABLE 3 Mean (±SEM) number of corpora lutea (CL), follicles >10 mm indiameter, number of cows with ≦2 CL at the time of ova/embryocollection, number of ova/embryos, fertilized ova and grades 1, 2, and 3embryos (transferable embryos) in beef cows treated with a single (60μg) or split-single (40-20 μg) I.M. injections of rFSH (TR55601) or 300mg Folltropin-V ® (Control) given in twice daily IM injections over 4days. Grades Grades 1, Follicle Cows with ≦2 Total Fertilized Grade 11&2 2, &3 No. “0” Treatment N CL >10 mm CL on Day 15 ova/embryos ovaembryos embryos embryos emb Control 10 14.1 ± 2.1 3.2 ± 1.1a 0 12.7 ±2.4 10.5 ± 1.6  7.5 ± 1.4 8.1 ± 1.4 9.0 ± 1.5 0 rFSH 60 μg 10 12.7 ± 2.8 4.6 ± 1.1ab 2 11.6 ± 3.0 9.1 ± 2.2 5.4 ± 1.2 6.6 ± 1.5 6.6 ± 1.5 2 rFSH40-20 μg 10 13.9 ± 1.5 8.5 ± 1.9b 0 11.2 ± 1.9 9.9 ± 1.8 6.2 ± 1.4 7.4 ±1.7 7.9 ± 1.7 0 P-value 0.6407 0.0262 0.1173 0.7317 0.6008 0.4339 0.60350.4462 0.1173

The superovulatory response and ova/embryo data for the second dosingare summarized in Tables 4 and 5. The mean number of CL, follicles >10mm and cows with <2 CL on the day of ova/embryo collection did notdiffer between groups. The mean number of total ova/embryos, fertilizedova and transferable embryos (Grades 1, 2 and 3) also did not differbetween groups.

Tables 4 and 5 show superovulation with single dose of TR55601 (rFSH).

TABLE 4 Mean (±SEM) number of CL, follicles > 10 mm in diameter andnumber of cows with ≦2 CL at the time of ova/embryo collection. Cowswere treated with a single (60 μg) I.M. injection of rFSH or 300 mgFolltropin-V (Control) given in twice daily I.M. injections over 4 d.Follicles > Cows with ≦2 Treatment N CL 10 mm CL on Day 15 Control 1012.7 ± 2.4 3.7 ± 0.9 0 rFSH 60 μg 15 14.7 ± 1.9 5.3 ± 1.3 1 P-value0.5730 0.3752 0.4047

TABLE 5 Mean (± SEM) number of ova/embryos, fertilized ova and grades 1,2, and 3 embryos (transferable embryos) in beef cows treated with asingle (60 μg) I.M. injection of rFSH (TR55601) or 300 mg Folltropin-V ® (Control) given in twice daily IM injections over 4 days. CowsGrades with Total Grades 1, 2 &3 “0” ova/ Fertilized Grade 1 1 & 2embryos transf. Treatment N embryos ova embryos embryos (transferable)emb. Control 10 11.9 ± 2.5 10.5 ± 2.2 3.2 ± 0.8 4.7 ± 1.1 4.9 ± 1.2 2rFSH 60 μg 14 13.4 ± 3.3 11.6 ± 3.0 3.5 ± 1.0 5.1 ± 1.5 6.1 ± 1.8 4P-value 0.8263 0.7958 0.8903 0.8608 0.9992 0.6326

For the final dosing experiment, only follicle data are available atthis time. There was no significant difference in follicle growthprofiles and the numbers of follicles on the day before inseminationbetween the rFSH and Folltropin-V groups. Ovulation and ova/embryo datawill be available shortly and will be presented separately forExperiment 3 and then combined with Experiments 1 and 2. Follicle datafor Experiment 3 are available and have been combined with that ofexperiments 1 and 2 and are presented in Table 6.

Table 6 shows three superovulations with TR55601 (rFSH) at 30 dayintervals.

TABLE 6 Mean (±SEM) number of CL, follicles >10 mm in diameter, numberof cows with ≦2 CL at the time of ova/embryo collection, number ofova/embryos, fertilized ova and grades 1, 2, and 3 embryos (transferableembryos) in beef cows treated with a single (60 μg) or split-single(40-20 μg) I.M. injections of rFSH (TR55601) or 300 mg Folltropin-V ®(Control) given twice daily IM injections over 4 days. Cows were treatedthree consecutive times at ~30 day intervals (3 experiments combined).Cows with Grades 1 Grades 1, 2 & 3 Cows with Follicles ≦2 CL on TotalFertilized Grade 1 & 2 embryos “0” transt. Experiments N CL >10 mm Day15 ova/embryos ova embryos embryos (transferable) emb Main EffectsExperiment 1 24 14.1 ± 1.4 5.0 ± 1.0 1 13.0 ± 1.8 11.0 ± 1.1 7.3 ±0.8^(a) 8.4 ± 0.9^(a) 8.9 ± 1.0^(a) 1 Experiment 2 24 13.7 ± 1.5 4.7 ±0.9 1 10.7 ± 1.8  8.9 ± 1.4 4.5 ± 1.0^(a) 5.6 ± 1.1^(ab) 6.6 ± 1.3^(ab)2 Experiment 3 24 15.5 ± 1.9 5.3 ± 1.0 3 12.8 ± 2.2 11.1 ± 1.9 3.4 ±0.7^(b) 5.0 ± 1.0^(b) 5.6 ± 1.2^(b) 6 P-value 0.9426 0.9196 0.42330.5881 0.5169 0.0031 0.0219 0.0480 0.0595 Treatments Control 30 13.9 ±1.2 3.7 ± 0.6^(a) 0 11.3 ± 1.5  9.7 ± 1.2 4.9 ± 0.8 6.0 ± 0.8 6.7 ± 1.02 rFSH 60 μg 42 14.8 ± 1.3 5.9 ± 0.8^(b) 5 12.8 ± 1.5 10.8 ± 1.3 5.2 ±0.7 6.8 ± 0.8 7.3 ± 0.9 7 P-value 0.9664 0.0224 0.0501 0.7893 0.91840.9391 0.9571 0.9840 0.2059 Experiment treatment 0.8844 0.7250 0.77200.8597 0.9385 0.9790 0.9769 interaction

The follicle characteristics from the beginning of treatment until justbefore artificial insemination are shown in Table 7 and FIGS. 6-9.

Tables 7 shows follicle numbers in superovulation model in nonlactatingcows. Follicle development (mean±SEM) was detected by ultrasonography ofbeef cows treated with 60 μg of rFSH given by single i.m. injection or300 mg Folltropin®-V given twice daily i.m. injections over 4 days.

TABLE 7 Day 0 Day 4 Day 8 Follicles Follicles Follicles ≧ Treatment N 3to 5 mm 3 to 5 mm 9 mm rFSH 18 13.2 ± 1.1 18.9 ± 1.7 14.7 ± 2.7Folltropin-V 18 12.4 ± 1.2 17.4 ± 1.3 18.4 ± 1.9 P-value 0.66 0.47 0.12

The numbers of follicles 3 to 5 mm in diameter did not differ amongtreatment groups (FIG. 6). Neither did the numbers of follicles 6 to 8mm in diameter (FIG. 7), follicles >9 mm (FIG. 8) or the mean folliclediameter over the days of treatment differ (FIG. 9).

Results obtained in this series of experiments can be interpreted tosuggest that the rFSH product induces a superovulatory response in beefcows that is not different from that of Folltropin-V. There is also noevidence of a decrease in superovulatory response as compared withFolltropin-V when cows are treated three times consecutively. Thesuperovulatory response in the final dosing would appear to be similarto the first two and this will be confirmed following ova/embryocollection. There is concern about the larger number of unovulated (>10mm) follicles in cows treated with rFSH (mainly due to high number ofunovulated follicles in two cows) that needs to be investigated further.More studies are also required to determine the optimal dosage of rFSHto superovulate beef and dairy cows and to determine the long termeffects of treating consecutively with rFSH more than three times.

Specificity of Insert for Improved Half-Life

To determine if the observed improved half-life is specific to thesequence of the insert of SEQ ID NO: 1, the human alpha subunit with theinsert was modified to remove the amino terminal valine. The two werethen tested for their ability to produce cAMP, along with the analogthat lacks the insert. The studies showed that the insert is sequencespecific and confers superior binding and half-life on the alpha subunit(see FIG. 9). The production conditions were then examined in variousgrowth conditions to optimize for maximal production (see Table 8).

Table 8 shows optimization of production of the human alpha subunit.

Average Interpolated Dilution Concentration Yields Samples OD ng/mlfactor ng/ml * dil ng/ml SEM ng/dish Insert 1 in GLP Sigma 6 1.175323.10155 800 18481.24 22783.156 4301.916 2506.1472 medium 0.9226516.92817 1600 27085.072 Insert 1 in Excell 302 1.1518 22.49597 80017996.776 20802.18 2805.404 2669.6131 medium 0.827 14.75474 160023607.584 Insert 1 in Lonza 1.1652 22.84037 800 18272.296 22269.6123997.316 2820.8175 medium 0.90043 16.41683 1600 26266.928 Insert 1 inHyclone 1.9913 24.68149 400 9872.596 12390.186 1140.7851 1734.6261medium 1.37845 13.94756 800 11158.048 0.9435 8.538053 1600 13660.8850.57165 4.64663 3200 14869.216

The insert was then examined along with the bovine counterpart and it isconfirmed that the increased half-life is specific to the sequence ofthe insert (see FIG. 11).

What is claimed:
 1. A modified bovine follicle-stimulating hormone (FSH)comprising SEQ ID NO: 2 with arginine, histidine, or lysinesubstitutions at K15, K17, K20, K24 and E18 and an insert of SEQ ID NO:1 between F6 and T7, wherein the modified bovine FSH has an increasedFSH receptor activation compared to bovine wild type FSH.
 2. Themodified bovine FSH of claim 1, wherein at least one substitution is anarginine substitution.
 3. The modified bovine FSH of claim 1, wherein atleast one substitution is a histidine substitution.
 4. The modifiedbovine FSH of claim 1, further comprising the beta subunit of a bovineFSH.
 5. The modified bovine FSH of claim 4, wherein at least onesubstitution is an arginine substitution.
 6. The modified bovine FSH ofclaim 5, wherein the substitution at E18 of SEQ ID NO: 2 is an argininesubstitution.
 7. The modified bovine FSH of claim 1, comprising aminoacid substitutions K15R, K17R, E18R, K20R and K24R, and the beta subunitof bovine FSH.
 8. The modified bovine FSH of claim 1 comprising an alphasubunit having a sequence of SEQ ID NO:
 7. 9. The modified bovine FSH ofclaim 8, further comprising the beta subunit of bovine FSH.
 10. Themodified bovine FSH of claim 1, wherein at least one substitution is alysine substitution.
 11. A modified bovine follicle-stimulating hormone(FSH) comprising SEQ ID NO: 2 with substitutions at K15R, K17R, K20R,K24R and E18 and an insert of SEQ ID NO: 1 between F6 and T7 and thebeta subunit of bovine FSH, wherein the modified bovine FSH has anincreased FSH receptor activation compared to wild type bovine FSH. 12.The modified bovine FSH of claim 11, wherein the alpha subunit comprisesSEQ ID NO:
 7. 13. The modified bovine FSH of claim 11, where the alphasubunit consists of SEQ ID NO:
 7. 14. The modified bovine FSH of claim1, wherein the modified bovine FSH has a longer half-life and durationof action compared to bovine wild-type FSH.